Cystinuria, a genetic disorder of cystine transport, is characterized by excessive excretion of L-cystine in the urine and recurrent cystine stones in the kidneys and, to a lesser extent, in the bladder. Current treatment of cystinuria has not changed over the last three decades. Patients are advised to drink substantial amounts of water (>4 liters/day) to reduce the concentration of free L-cystine in urine and to alkalinize the urine to increase cystine solubility, advice that challenges patient adherence. Drug therapy in severe conditions is limited to two thiol drugs that react with L-cystine to form more soluble mixed disulfides, but these drugs have poor patient compliance due to adverse reactions that, in some cases, may be life-threatening. Thus, there is a need to reduce or eliminate the risks associated with therapy for cystinuria. In this application, we propose a new approach to prevent recurrent cystine stone formation in patients with cystinuria. Our substantive in vitro studies indicate that L-cystine diamides are potent inhibitors of cystine crystallization, which is a required condition for stone formation. Our preliminary in vivo studies indicate that one compound (LH708) from this family is effective in preventing L-cystine stone formation in Slc3a1 knockout mice, which we generated in our laboratories as a model for cystinuria. Building on our in vitro and in vivo data, we will design, synthesize, and evaluate new analogs and prodrugs of L-cystine diamides with sufficient metabolic stability and oral bioavailability that can effectively inhibit L-cystine crystallization in vivo and thus prevent L-cystine stone formation. The effectiveness of these new compounds to inhibit L-cystine crystallization will be tested using a combination of in vitro fluorescence-based solubility and real-time in situ crystal growth inhibition assays. The most effective inhibitors then will be evaluated for their in vivo efficacy, safety, and bioavailability in Slc3a1 knockout mice and for their preclinical ADME-Tox properties. The combined results from these studies will enable the selection of one top drug candidate and one backup candidate for IND-directed preclinical evaluations, for which we will seek assistance from the NIH NCATS program.